Nutritional composition for infants

ABSTRACT

A nutritional composition for infants comprises a protein source, a lipid source and a carbohydrate source wherein the lipid source includes at least 16 wt % linoleic acid and at least 2 wt % α-linolenic acid expressed as a percentage of total fatty acid content in each case and in amounts such that the ratio of linoleic acid:α-linolenic acid is in the range from 1 to 10.

This invention relates to a nutritional composition, more specificallyto a nutritional composition for infants which may help to reduce therisk of development of obesity later in life.

Mother's milk is recommended for all infants. However, in some casesbreast feeding is inadequate or unsuccessful or inadvisable for medicalreasons or the mother chooses not to breast feed either at all or for aperiod of more than a few weeks. Infant formulas have been developed forthese situations.

The prevalence of obesity and overweight in adults, children andadolescents has increased rapidly over the past 30 years in the UnitedStates and globally and continues to rise. Childhood overweight andobesity currently affects 18 million children under age 5 worldwide.Almost 30% of US children and adolescents and between 10 and 30% ofEuropean children are overweight or obese.

Increasingly it is believed that the first 6 months of life representone of the most important postnatal periods for human fat massdevelopment and consequently may be a critical window for programmingexcess of adiposity later in life. Moreover, human epidemiological dataand animal studies evidence that elevated body weight at birth or duringinfancy are associated with a risk for development of diseases such asmetabolic syndrome, Type 2 diabetes and cardiovascular problems later inlife.

Korotkova et al. investigated the influence of the ratio of linoleicacid (LA) to α-linolenic acid (ALA) in the maternal diet on serum leptinlevels in their suckling pups and found that feeding dams a diet rich inALA (LA/ALA ratio 0.42) decreased serum leptin levels in the sucklingpups compared with pups whose mothers were fed a diet containing both LAand ALA (LA/ALA ratio 9.0). Mean serum leptin levels of a third group ofpups whose mothers had been fed a diet rich in LA (LA/ALA ratio 216)were between the levels in the other two groups but not significantlydifferent from either. The authors chose to study serum leptin levels inthe pups as a number of earlier studies already suggest that circulatingleptin levels during the perinatal period could be important for normaldevelopment and health. On the basis of their findings, they go on topredict that it could be the balance between LA and ALA in the maternaldiet rather than the amount of LA or ALA per se that is important foradipose tissue growth (Korotkova et al, “Leptin levels in rat offspringare modified by the ratio of linoleic to α-linolenic acid in thematernal diet”, Journal of Lipid Research, Vol 43, 2002, pages1743-1749).

In WO2008/054192, it is claimed that the whole adipose tissue mass ofinfants is not a good predictor to determine the risks of diseases laterin life and that it is rather the accumulation of visceral fat mass inearly infancy that should be considered. It has been demonstrated thatvisceral adipocyte count is primarily determined during infancy and theinventors of WO2008/054192 believe that it would be useful to be able tocontrol visceral adipogenesis during this period. The solution proposedin WO2008/054192 is to reduce the amount of LA fed to infants such thatthe LA/ALA ratio is between 2 and 6 whilst at the same time ensuringthat LA amounts to less than 14.5% by weight of total fatty acids.

However, this approach may have practical limitations as regardsnutrition of human infants given that both LA and ALA are essentialfatty acids, that is, fatty acids which cannot be synthesized by thebody.

There is, therefore, clearly a need for alternative nutritionalapproaches aimed at reducing accumulation of fat mass during childhoodand thus reducing risk of developing obesity later in life as well asthe risk of developing diseases associated with such obesity.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that feeding a compositionrelatively rich in α-linolenic acid during infancy reduces accumulationof total fat mass later in life.

Accordingly, the present invention provides a nutritional compositioncomprising a protein source, a lipid source and a carbohydrate sourcewherein the lipid source includes at least 16 wt % linoleic acid and atleast 2 wt % α-linolenic acid expressed as a percentage of total fattyacid content in each case and in amounts such that the ratio of linoleicacid:α-linolenic acid is in the range from 1 to 10.

The invention also extends to the use of linoleic acid and α-linolenicacid in the manufacture of a nutritional composition for administrationto an infant in the first six months of life of the infant so as toreduce the risk of development of obesity later in life wherein thecomposition includes at least 16 wt % linoleic acid and at least 2 wt %α-linolenic acid expressed as a percentage of total fatty acid contentof the composition in each case and in amounts such that the ratio oflinoleic acid:α-linolenic acid is in the range from 1 to 10.

The invention further extends to a method of reducing the risk that aninfant will develop obesity later in life comprising feeding to theinfant in the six months of its life a nutritional compositioncomprising a protein source, a lipid source and a carbohydrate sourcewherein the lipid source includes at least 16 wt % linoleic acid and atleast 2 wt % α-linolenic acid expressed as a percentage of total fattyacid content in each case and in amounts such that the ratio of linoleicacid:α-linolenic acid is in the range from 1 to 10.

Without wishing to be bound by theory, the present inventors believe onthe basis of their experimental observations that feeding a diet rich inα-linolenic acid during infancy may in some way reduce either or both ofadipogenesis and increase in size of adipocytes and that thesebeneficial effects may persist in later life, thus reducing the risk ofdevelopment of obesity later in life. Further, obesity is known to beassociated with increased risk of conditions such as insulin resistanceand glucose intolerance which may lead to development of Type 2diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the evolution of body weight of three groups of guinea pigpups fed different diets over the first 136 days of life.

FIG. 2 shows the median body weight gain of pups in the three groupsbetween day 21 and day 79.

FIG. 3 shows the number of large adipocytes plotted as a function of ALAintake.

FIG. 4 shows the evolution of fat mass in the three groups expressed ingrams from day 21 to day 128.

FIG. 5 shows the evolution of fat mass in the three groups expressed asa percentage of total body weight at day 79, day 107 and day 128.

FIG. 6 shows the mean plasma insulin levels of the three groups at day136.

FIG. 7 shows the pancreatic insulin contents of the three groups at day136.

DETAILED DESCRIPTION OF THE INVENTION

In this specification, the following expressions have the meaningsassigned to them below:—

“ALA” means α-linolenic acid (C18:3n-3);

“infant” means a child under the age of 12 months and “infancy” shall beconstrued accordingly; For the purpose of the present document the term“infant” comprises young baby animals, such as young pets, young dogs,young horses or young cats.

“LA” means linoleic acid (C18:2n-6);

“later in life” means, in the context of an infant, any point in lifeafter completion of infancy. One example of such a point would be at theage of the adiposity rebound which, in human infants, typically occursbetween the ages of five and six years.

All percentages and ratios are by weight unless otherwise specified.

A nutritional composition according to the present invention comprisesat least 16% LA and at least 2% ALA. Preferably the LA content is in therange from 18 to 30% of total fatty acids, more preferably 18 to 25%.Preferably the ALA content is in the range from 2 to 12% of total fattyacids, more preferably from 6 to 11%. The LA:ALA ratio is preferably inthe range from 2 to 6.

Preferably, the nutritional composition of the present invention has aprotein content of less than 1.85 g/100 kcal. The detailed make-up ofthe protein source is not believed to be critical to the presentinvention provided that the minimum requirements for essential aminoacid content are met and satisfactory growth is ensured. Thus, proteinsources based on cows' milk proteins such as whey, casein and mixturesthereof may be used as well as protein sources based on soy. However,mixtures of whey and casein proteins are preferred. The casein:wheyratio may lie in the range from 70:30 to 30:70 but is preferably 30:70.

The protein(s) in the protein source may be intact or partiallyhydrolysed or a mixture of intact and hydrolysed proteins may be used.The protein source may additionally be supplemented with free aminoacids if this is necessary to meet the minimum requirements foressential amino acid content. These requirements are published forexample in EC Directive 2006/141/EC.

As noted above, the preferred protein source is a mixture of casein andwhey proteins. The whey protein may be a whey protein isolate, acidwhey, sweet whey or sweet whey from which the caseino-glycomacropeptidehas been removed (modified sweet whey).

Preferably, however, the whey protein is modified sweet whey. Sweet wheyis a readily available by-product of cheese making and is frequentlyused in the manufacture of nutritional compositions based on cows' milk.However, sweet whey includes a component which is undesirably rich inthreonine and poor in tryptophan called caseino-glycomacropeptide(CGMP). Removal of the CGMP from sweet whey results in a protein with athreonine content closer to that of human milk. A process for removingCGMP from sweet whey is described in EP 880902.

If modified sweet whey is used as the whey protein in a mixture of 70%whey and 30% casein, the protein source may be supplemented by freehistidine in an amount between 0.1 and 0.3% of total protein content.

The nutritional composition of the present invention contains a sourceof carbohydrates. The preferred source of carbohydrates is lactosealthough other carbohydrates such as saccharose, maltodextrin, andstarch may also be added. Preferably, the carbohydrate content of thenutritional composition is between 9 and 14 g/100 kcal.

The nutritional composition of the present invention contains a sourceof lipids. In addition to LA and ALA, the lipid source may include anylipid or fat which is suitable for use in nutritional compositions to befed to infants. Preferred fat sources include coconut oil, low erucicrapeseed oil (canola oil), soy lecithin, palm olein, and sunflower oil.The lipid source may also include small amounts of preformed long chainpolyunsaturated fatty acids arachidonic acid (C20:4n-6) anddocosahexaenoic acid (C22:6n-3). Fish oils are a suitable source ofdocosahexaenoic acid, alternatively single cell microbial oils aresuitable sources of both arachidonic acid and docosahexaenoic acid. Intotal, the lipid content may be between 4.4 and 6 g/100 kcal. The ratioof arachidonic acid:docosahexaenoic acid in the lipid source ispreferably between 2:1 and 1:1.

The nutritional composition may also contain all vitamins and mineralsunderstood to be essential in the daily diet in nutritionallysignificant amounts. Minimum requirements have been established forcertain vitamins and minerals. Examples of minerals, vitamins and othernutrients optionally present in the nutritional composition includevitamin A, vitamin B₁, vitamin B₂, vitamin B₆, vitamin B₁₂, vitamin E,vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin,pantothenic acid, choline, calcium, phosphorous, iodine, iron,magnesium, copper, zinc, manganese, chloride, potassium, sodium,selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals areusually added in salt form.

If necessary, the nutritional composition may contain emulsifiers andstabilisers such as soy lecithin, citric acid esters of mono- anddi-glycerides, and the like. This is especially the case if thecomposition is provided in liquid form.

The nutritional composition may optionally contain other substanceswhich may have a beneficial effect such as probiotic bacteria, fibres,lactoferrin, nucleotides, nucleosides, and the like in the amountscustomarily found in nutritional compositions to be fed to infants.

The nutritional composition may be prepared in any suitable manner. Forexample, a nutritional composition may be prepared by blending togetherthe protein source, the carbohydrate source, and the lipid source inappropriate proportions. If used, emulsifiers may be included in theblend at this stage. The vitamins and minerals may be added at thispoint but are usually added later to avoid thermal degradation. Anylipophilic vitamins, emulsifiers and the like may be dissolved into thefat source prior to blending. Water, preferably water which has beensubjected to reverse osmosis, may then be mixed in to form a liquidmixture.

The liquid mixture may then be thermally treated to reduce bacterialloads. For example, the liquid mixture may be rapidly heated to atemperature in the range of about 80° C. to about 110° C. for about 5seconds to about 5 minutes. This may be carried out by steam injectionor by heat exchanger; for example a plate heat exchanger.

The liquid mixture may then be cooled to about 60° C. to about 85° C.;for example by flash cooling. The liquid mixture may then behomogenised; for example in two stages at about 7 MPa to about 40 MPa inthe first stage and about 2 MPa to about 14 MPa in the second stage. Thehomogenised mixture may then be further cooled and any heat sensitivecomponents; such as vitamins and minerals may be added. The pH andsolids content of the homogenised mixture is conveniently standardisedat this point.

If it is desired to produce a powdered composition, the homogenisedmixture is transferred to a suitable drying apparatus such as a spraydrier or freeze drier and converted to powder. The powder should have amoisture content of less than about 5% by weight.

If it is desired to produce a liquid composition, the homogenisedmixture is filled into suitable containers; preferably aseptically.However, the liquid composition may also be retorted in the container.Suitable apparatus for carrying out filling of this nature iscommercially available. The liquid composition may be in the form of aready to feed composition having a solids content of about 10 to about14% by weight or may be in the form of a concentrate; usually of solidscontent of about 20 to about 26% by weight.

The invention will now be further illustrated by reference to thefollowing examples.

Example 1

An example of the composition of a nutritional composition according tothe invention is given below:—

Nutrient per 100 kcal per litre Energy (kcal) 100 630 Protein (g) 1.59.45 (skimmed milk powder, modified sweet whey) free histidine (mg) 2.515.8 casein:whey ratio 40:60 40:60 Fat (g) 5.3 33.4 Linoleic acid (g)1.0 6.6 α-Linolenic acid (g) 0.5 3.3 DHA (mg) 11.5 72.5 ARA (mg) 11.572.5 Linoleic acid: α-Linolenic acid 2 2 Lactose (g) 11.6 73.1 Mineralsand Electrolytes Na (mg) 25 158 K (mg) 89 561 Cl (mg) 64 403 Ca (mg) 64403 P (mg) 32 202 Ca/P 2.0 2.0 Mg (mg) 6.9 43.5 Mn (μg) 8.0 50.4Vitamins and Trace Elements Vitamin A (IU) 350 2205 Vitamin D (IU) 60378 Vitamin E (IU) 1.2 7.6 Vitamin K1 (μg) 8.0 50.4 Vitamin C (mg) 10 63Vitamin B1 (mg) 0.07 0.44 Vitamin B2 (mg) 0.15 0.95 Niacin (mg) 1.0 6.3Vitamin B6 (mg) 0.075 0.47 Folic acid (μg) 12 75.6 Pantothenic acid (mg)0.45 2.83 Vitamin B12 (μg) 0.3 1.89 Biotin (μg) 2.2 13.9 Choline (mg) 1063 Inositol (mg) 5.0 31.5 Taurine (mg) 7.0 44.1 Carnitine (mg) 1.6 10.1Fe (mg) 1.2 7.56 I (μg) 15 94.5 Cu (mg) 0.07 0.44 Se (μg) 2.0 12.6 Zn(mg) 0.75 4.72 Nucleotides CMP (mg) 2.3 14.5 UMP (mg) 1.5 9.5 AMP (mg)0.7 4.4 GMP (mg) 0.3 1.9

A nutritional composition according to the invention may be fed to aninfant as the sole source of nutrition from birth to the age of four tosix months and subsequently as part of a mixed diet during theintroduction of solid foods until weaning is complete at about the ageof 12 months.

Example 2

This example investigates the effect of feeding diets containingdifferent amounts of LA and ALA to newborn guinea pig pups on thedevelopment of total fat mass of the pups in the post-weaning period.The infant guinea pig is considered to be a good animal model to studyto predict the development fat mass in human infants because, likenewborn human infants, newborn guinea pig pups are born with anappreciable amount of body fat whilst newborn rat pups are very lean.

Study Design:

Newborn male guinea pigs were divided into three groups with 20 animalsin each group. Each group was fed a suckling/weaning diet in which 44%of the energy was supplied by fat for 21 days. The different diets wereisocaloric and differed only in their ALA contents. The high ALA dietcontained 10% ALA based on total fatty acids, the medium ALA dietcontained 2.4% ALA based on total fatty acids and the low diet contained0.85% ALA based on total fatty acids. The levels of LA were keptrelatively constant between diets such that the LA:ALA ratios were about2, 10 and 30 respectively. Further details of the fatty acid compositionof the three diets are given in Table 1.

TABLE 1 Fatty acid composition of the suckling/weaning diets: % of totalfatty acids 10% ALA 2.4% ALA 0.8% ALA C12:0 7.1 8.2 7.9 C14:0 3.4 3.93.7 C16:0 22.7 22.0 21.8 C16:1 0.2 0.1 0.1 C18:0 3.0 3.2 3.0 C18:1 33.533.0 33.3 C18:2n-6 (LA) 20.2 26.2 28.5 C18:3n-3 (ALA) 9.9 2.4 0.8 C20:00.0 0.0 0.0 C20:1 0.0 0.0 0.0 C20:4n-6 0.0 0.0 0.0 C20:5n-3 0.0 0.0 0.0C22:0 0.0 0.0 0.0 C22:5n-3 0.0 0.0 0.0 C22:6n-3 0.0 0.0 0.0 LA/ALA 2.010.7 33.6

At the end of the suckling/weaning period (day 21), all groups were feda diet with a moderately high fat content (35% of energy from fat)containing 2% ALA and 26% LA until day 136. Body weight and fat masswere recorded at days 21, 51, 79, 107, 128 and 136 and retroperitonealadipose tissue cellularity, plasma fatty acid composition, and insulinwere recorded at day 21 and day 136.

Results:

As expected, at the end of the suckling/weaning period (day 21) theconcentrations of ALA, in plasma phospholipid (Table 2) and triglyceride(Table 3) fractions, were higher in the 10% ALA group than in the othergroups.

TABLE 2 Fatty acid (μg/ml) composition in plasma phospholipids at d 21.High ALA Medium ALA Low ALA C10:0  0.1 ± 0.02  0.1 ± 0.03  0.1 ± 0.07C12:0  0.2 ± 0.03  0.2 ± 0.02  0.3 ± 0.13 C14:0  0.8 ± 0.23  1.0 ± 0.10 1.1 ± 0.17 C16:0 35.9 ± 9.1  49.3 ± 8.9  40.2 ± 3.6  C17:0  1.2 ± 0.38 1.9 ± 0.41  1.2 ± 0.10 C18:0 70.1 ± 23.6 98.6 ± 28.1 68.6 ± 4.2 C18:1n-9 cis + trans 23.4 ± 7.6  30.8 ± 7.3  23.2 ± 1.9  C18:1n-7 cis +trans 3.1 ± 0.8 6.2 ± 1.4 5.2 ± 0.9 C18:2n-6 (LA) 67.5 ± 22.2 99.0 ±26.3 77.0 ± 7.5  C18:3n-6 (GLA)  0.1 ± 0.06  0.1 ± 0.09  0.2 ± 0.13C18:3n-3 (ALA) 2.9 ± 1.8 0.9 ± 0.2 0.6 ± 0.2 C20:0 1.2 ± 0.5 1.5 ± 0.2 0.9 ± 0.03 C20:1n-9 0.5 ± 0.2  0.7 ± 0.08 0.6 ± 0.1 C20:2n-6 0.8 ± 0.11.2 ± 0.1 1.3 ± 0.2 C20:3n-6 0.9 ± 0.2  1.1 ± 0.25  1.0 ± 0.07 C20:4n-6(AA) 11.4 ± 3.6  14.8 ± 3.0  11.7 ± 1.1  C20:3n-3  0.3 ± 0.08  0.2 ±0.06  0.1 ± 0.03 C22:0 1.9 ± 0.9 2.1 ± 0.4 1.1 ± 0.2 C22:1n-9 0.4 ± 0.20.4 ± 0.1 1.5 ± 1.0 C20:5n-3 (EPA) 0.2 ± 0.1  0.1 ± 0.03  0.1 ± 0.03C22:2n-6 1.1 ± 0.5 1.5 ± 0.2 0.8 ± 0.1 C22:4n-6 1.2 ± 0.5 1.8 ± 0.3 1.2± 0.2 C24:0 2.6 ± 1.2 3.4 ± 0.6 1.9 ± 0.4 C24:1n-9 2.4 ± 0.8 2.8 ± 0.32.0 ± 0.5 C22:5n-3 (DPA) 2.1 ± 0.8 1.7 ± 0.3 0.9 ± 0.1 C22:6n-3 (DHA)1.1 ± 0.4 1.3 ± 0.3 0.7 ± 0.1 Mean ± SEM

TABLE 3 FA (μg/ml) composition in plasma triglycerides at d 21. High ALAMedium ALA Low ALA C10:0 0.2 ± 0.1  0.1 ± 0.04  0.2 ± 0.01 C12:0 0.9 ±0.2 1.0 ± 0.1 0.8 ± 0.3 C14:0 4.0 ± 1.1 4.6 ± 0.7 4.0 ± 2.4 C16:0 49.1 ±10.7 52.8 ± 5.7  42.2 ± 18  C17:0 0.9 ± 0.2 1.1 ± 0.2  0.7 ± 0.09 C18:015.7 ± 3.3  14.4 ± 2.3  10.5 ± 3.5  C18:1 n-9 cis 85.0 ± 17.8 85.4 ±6.0  63.2 ± 18  C18:1 n-7 cis 2.6 ± 0.5 2.8 ± 0.3 2.0 ± 0.6 C18:2 n-6(LA) 72.8 ± 13.7 85.4 ± 6.0  71.1 ± 27.9 C18:3n-6 (GLA) 0.2 ± 0.2  0.6 ±0.05 0.5 ± 0.1 C18:3n-3 (ALA)  11.1 ± 2.5 ^(a)   5.1 ± 0.7 ^(b)   2.7 ±0.2 ^(b) C20:0 0.5 ± 0.2 0.7 ± 0.1 0.2 ± 0.1 C20:1n-9 1.2 ± 0.1 1.3 ±0.1 1.1 ± 0.5 C20:2n-6 1.4 ± 0.2 1.5 ± 0.2 1.6 ± 0.8 C20:3n-6 0.8 ± 0.1 0.7 ± 0.09  0.6 ± 0.09 C20:4n-6 (AA) 3.7 ± 0.4 3.5 ± 0.3 3.7 ± 0.3C20:3n-3 0.4 ± 0.1  0.2 ± 0.05  0.1 ± 0.09 C22:0  0.4 ± 0.04  0.4 ± 0.05 0.3 ± 0.02 C22:1n-9  0.3 ± 0.08  0.2 ± 0.02  0.2 ± 0.03 C20:5n-3 (EPA) 0.3 ± 0.04  0.1 ± 0.04  0.1 ± 0.08 C22:2n-6  0.3 ± 0.04  0.3 ± 0.06 0.2 ± 0.08 C22:4n-6 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.2 C24:0 0.7 ± 0.1 0.8 ±0.1 0.6 ± 0.1 C22:5n-3 (DPA) 0.8 ± 0.1 0.6 ± 0.1  0.4 ± 0.01 C22:6n-3(DHA) 0.3 ± 0.1  0.3 ± 0.06  0.2 ± 0.05 Mean ± SEM Different lettersindicate statistical significance at P < 0.05, ND; not detectable

As may be seen from FIG. 1, mean body weight did not differsignificantly between the three groups during the course of theexperiment. However, FIG. 2 shows that between day 21 and day 78 thehigh ALA group tended to gain less body weight than the other groups.

At the end of the suckling/weaning period (day 21), total fat massmeasured by NMR did not differ between the groups. Nevertheless, onaverage, the diameter of adipocyte cells from retroperitoneal adiposetissue of the high ALA group tended to be smaller than in the othergroups (Table 4).

TABLE 4 Adipocyte diameter. Groups Cell diameter (μm) High ALA 27.5 ±0.8 Medium ALA 31.1 ± 1.5 Low ALA 31.9 ± 1.7 Mean ± SEM

The High ALA group had a smaller number of adipocytes in the largerdiameter range (from 30 to 50 μm) (Table 5).

TABLE 5 Total cell number (×10⁶) per diameter range and per g ofretroperitoneal adipose tissue at d 21. Cell diameter 10% ALA 2.4% ALA0.8% ALA 15-19 1.27 ± 0.2 1.19 ± 0.2 0.91 ± 0.2 19-29 3.82 ± 0.6 3.70 ±0.5 3.68 ± 0.8 30-39 2.18 ± 0.4 3.54 ± 0.2 4.43 ± 0.8 40-49 0.37 ± 0.11.47 ± 0.2 1.85 ± 0.7 50-60  0.07 ± 0.02  0.34 ± 0.04 0.49 ± 0.3 Mean ±SEM.

Interestingly, a dose response relationship was observed between thenumber of large adipocytes (>40 μm) and the percentage of ALA in thediet. As may be seen from FIG. 3, the higher was the ALA intake, thelower was the number of large adipocytes.

Surprisingly and as may be seen from FIGS. 4 and 5, the Low ALA grouphad higher fat mass values in both gram and percentage values than theHigh ALA group at day 79 (p<0.01), day 107 (p<0.01) and day 128(p=0.08).

Plasma insulin levels were not different between groups at day 21.However, as shown in FIG. 6, at the end of the experiment, the Low ALAgroup showed about a 1.5-fold higher concentrations of plasma insulincompared to the other two groups. FIG. 7 shows that similar results wereobserved regarding the pancreatic insulin content. The Low ALA groupshowed about a 1.4-fold increase in the pancreatic insulin content whencompared to the High ALA group (p=0.08).

This example clearly demonstrates that feeding a diet rich in ALA duringinfancy plays an important role in programming or imprinting the adiposetissue in such a way as to reduce its susceptibility to excessivedevelopment later in life. Specifically, the results indicate that a lowALA intake with high LA:ALA ratio during the suckling/weaning periodleads to increased adiposity and a tendency to hyperinsulinemia later inlife.

Example 3

This example investigates the effect of feeding diets containingdifferent amounts of LA and ALA to newborn guinea pig pups on theproliferation rates of adipose tissue cells (AT-cells) and on de novolipogenesis (DNL) using the D₂O method. The High ALA group and the LowALA group from Example 2 were investigated. Ten pups in each groupreceived D₂O for 5 days prior sacrifice at day 21. The other 10 pupsreceived D₂O for 5 days prior sacrifice at day 136. The deuterated waterwas administered intraperitoneally (sterilized 0.9% NaCl, 99% D₂O at 35mg/g of body weight) in the morning of the first day. Thereafter, thepups drank deuterated water (8% enriched in deuterium) ad libitum forthe remainder of the 5 days. They were then sacrificed by exsanguinationunder isoflurane anesthesia. The blood was collected from the aorta inheparinized vials (minimum of 200 μL). The retroperitoneal, epididymaland subcutaneous adipose tissues and the liver were collected and aswell as the bone marrow from the rear-limb and kept at −80° C. untilanalysis.

The analytical procedure has been previously detailed (Neese R A, SilerS Q, Cesar D, Antelo F, Lee D, Misell L, Patel K, Tehrani S, Shah P,Hellerstein M K, “Advances in the stable isotope mass spectrometricmeasurement of DNA synthesis and cell proliferation” Analy Biochem 2001;298: 189-195). Briefly, the genomic DNA of AT-cells and thetriglycerides (TG) were extracted from the adipose tissues (about 50mg). Deuterium enrichment in DNA and palmitate from TG were determinedby mass-spectrometry hyphenated with gas-chromatography (GC/MS,Hewlett-Packard, Palo Alto, Calif.).

Use of D₂O incorporation for the calculation of DNA replication (cellproliferation) and DNL is based on the precursor-product relationship(Hellerstein M K, Neese R. “Mass isotopomer data analysis: a techniquefor measuring biosynthesis and turnover of polymers” Am J Physiol 1992;263: E988-E1001). In this method the cell divisions that occurred duringthe labeling period are counted by quantifying the proportion of labeledDNA strands present. The fractional proliferation rate of adipose tissuecells (FSRcell in % new cell/5 days) was calculated as the EM1 (isotopicenrichment in excess) in adipose DNA divided (normalized) by EM1 in bonemarrow DNA. Bone marrow enrichment was used to approximate maximum DNAenrichment achievable within each animal.

DNL or “fractional synthesis rate of palmitate” (FSRpalmitate in % newpalmitate/5 days) in TG was calculated from the label deuteriumincorporation from water into TG-palmitate using mass isotopomerdistribution analysis (MIDA). The present maximum deuterium enrichmentreachable in palmitate was calculated based upon the measured body waterenrichment (calculations are detailed in Hellerstein M K and Neese R A“Mass isotopomer distribution analysis at eight years: theoretical,analytic, and experimental considerations” Am J Physiol 1999; 276:E1146-E1170).

Results

The adipose tissue and liver weights are summarized in Table 6.

TABLE 6 Adipose tissue (AT) and liver weights (in g) at day 21 and day136. Groups Liver Epididymal AT* Retroperitoneal AT d 21  High ALA 8.57± 0.54 0.15 ± 0.02 0.56 ± 0.14 Low ALA 7.09 ± 0.40 0.09 ± 0.01 0.38 ±0.06 d 136 High ALA 29.73 ± 2.15     8.09 ± 1.55 ^(a) 9.36 ± 1.21 LowALA 30.10 ± 1.11   10.94 ± 0.58 ^(b) 10.98 ± 0.54  Different lettersindicate statistical significance at P < 0.05. *At d 136, P = 0.020between High ALA/Low

The effect of early diet treatment on the fractional synthesis rate (FSRor fractional proliferation rate) of cells in adipose depot issummarized in Table 7. The AT-cells include all type of cells such asthe adipocytes and the stroma-vascular cells (pre-adipocytes,enterocytes, macrophages, etc.) of the whole adipose tissue.

TABLE 7 FSR (in % new cells/5 days) at d 21 and d 136 GroupsSubcutaneous AT* Epididymal AT Retroperitoneal AT d 21  High ALA 8.9 ±8.7 13.5 ± 2.0 5.3 ± 2.3 Low ALA 18.7 ± 13.7 15.0 ± 4.1 3.3 ± 0.7 d 136High ALA   8.6 ± 1.7^(a)  7.7 ± 1.3 4.0 ± 0.6 Low ALA 15.2 ± 1.3^(b) 7.8 ± 2.6 7.9 ± 2.3 Different letters indicate statistical significanceat P < 0.05. *At d 136, P = 0.021 between High ALA/Low ALA

These results show a higher (doubled) cell proliferation rate in thesubcutaneous adipose tissue at day 136 (P=0.021) and a higher cellproliferation (but not significant) in the retroperitoneal and anunchanged cell proliferation in the epididymal adipose tissues in theLow ALA group compared with High ALA group. Since the subcutaneousadipose tissue is the major fat depot in the whole body of adultguinea-pigs, the increase of cell proliferation in this adipose tissuemay explain the increased fat mass composition observed after a low ALAintake (results obtained in Example 2). Mechanistically, the resultssuggest that the increase in body fat mass in the Low ALA group involvesan increase in the adipose tissue cell proliferation rate (ahyperplasia) in the guinea-pigs at the later adult period.

A low ALA intake did not induce changes in de novo lipogenesis (DNL) inthe fat deposits but a higher hepatic DNL (P<0.001) was observed in thelow ALA group as compared to the high ALA group at day 21 (Table 8).

TABLE 8 DNL (in % new palmitate/5 days) at day 21 and day 136 GroupsLiver Subcutaneous AT Epididymal AT Retroperitoneal AT d 21  High ALA  8.8 ± 2.6^(a) 9.3 ± 6.6 14.9 ± 2.8  13.8 ± 5.4  Low ALA 17.4 ± 3.7^(b)2.2 ± 1.8 17.1 ± 2.5  12.9 ± 2.3  d 136 High ALA 5.7 ± 0.6 1.87 ± 0.261.18 ± 0.16 1.40 ± 0.39 Low ALA 5.6 ± 0.4 1.29 ± 0.18 0.92 ± 0.12 1.06 ±0.07 Different letters indicate statistical significance at P < 0.001.

This example 3 shows that a low ALA intake during the suckling/weaningperiods increases the rate of hepatic DNL in 21 days-old guinea pigs andincreases the cell proliferation rate in the subcutaneous adipose tissuein adult guinea pigs.

The invention claimed is:
 1. A nutritional composition comprising 4.4 to6 g/100 kcal of the composition of a lipid source, 9 to 14 g/100 kcal ofthe composition of a carbohydrate source, and less than 1.85 g/100 kcalof the composition of a protein source wherein the lipid sourcecomprises at least 16 wt % linoleic acid and 6 to 11 wt % α-linolenicacid expressed as a percentage of total fatty acid content in each caseand the ratio of linoleic acid:α-linolenic acid is from 1 to 10, and thecomposition comprises arachidonic acid (ARA) and docosahexaenoic acid(DHA) in a ratio of 2:1 to 1:1 ARA:DHA.
 2. A nutritional compositionaccording to claim 1, wherein the linoleic acid content is from 18 to30% of total fatty acids.
 3. A nutritional composition according toclaim 1, wherein the linoleic acid content is from 18 to 25% of totalfatty acids.
 4. A nutritional composition according to claim 1, whereinthe α-linolenic acid content is from 2 to 12% of total fatty acids.
 5. Anutritional composition according to claim 1, wherein the α-linolenicacid content is 10 wt % of total fatty acids.
 6. A nutritionalcomposition according to claim 1, wherein the linoleic acid:α-linolenicacid ratio is from 2 to 6.